The motor dysfunction resulting from spinal cord injury (SCI) is precipitated by the neural insult and is exacerbated by other factors that hinder motoneuron survival and muscle recovery, including disuse and low testosterone (T). The success of bodyweight-supported treadmill training (BWSTT) diminishes as the SCI severity worsens. We have developed a novel strategy involving BWSTT with adjuvant T-enanthate (TE) drug treatment that promotes use-dependent neuroplasticity, in-part, by preserving white matter at the spinal lesion and by supporting motoneuron survival, which stimulates neuromuscular recovery. However, the supraphysiologic TE dose we used produced prostate enlargement in our rodent severe contusion SCI model, which limits translation. The purpose of this proposal is to improve the translational applicability of BWSTT+TE by identifying the lowest TE dose that enhances BWSTT-mediated neuromotor improvement, in an effort to limit prostate growth and other androgenic side-effects. Secondly, we will determine the influence of estradiol (E2) on BWSTT-mediated locomotor recovery and neuromuscular plasticity. The latter remains important from translational and mechanistic perspectives because (1) T is converted to E2, via aromatase, within the central nervous system and (2) E2 treatment produces potent neuroprotection in rats after SCI. To provide comprehensive evidence of neuroplasticity, we will evaluate functional adaptations in our rodent SCI model in response to the proposed treatments and assess anatomical changes that occur at the spinal cord lesion and distal to the lesion, and in spinal motoneurons and muscle. To support this proposal, we have established a male rodent severe mid-thoracic contusion SCI model that exhibits persistent hindlimb paralysis and progressive muscle decline and 50% lower circulating T than non-SCI animals, similar to the T deficiency present in nearly all men after SCI. Our data indicate that BWSTT+TE restored hindlimb overground walking after severe SCI more so than BWSTT (alone) or TE (alone). Moreover, BWSTT+TE improved recovery of muscle fiber cross-sectional area (fCSA), muscle force output, and prevented the deleterious slow (oxidative) to fast (glycolytic) fiber-type transition in muscle. In Aim 1, we will perform a dose-optimization experiment to identify the lowest effective TE dose and we will build upon our original data by comprehensively evaluating locomotor recovery and neuromuscular plasticity in response to BWSTT+TE. The primary outcomes are open- field locomotor recovery, soleus muscle function, soleus fCSA and fiber type distribution. In Experiment 1a, SCI animals will remain untreated (vehicle) or will receive BWSTT with low, moderate, or high-dose TE, with the lowest effective dose advancing. In Experiment 1b, SCI animals will receive vehicle, BWSTT, TE, or BWSTT+TE. Aim 2 will then determine the influence of estradiol (E2) on BWSTT-mediated locomotor recovery and neuromuscular plasticity. In Experiment 2a, SCI rats will receive vehicle, BWSTT, BWSTT+AN (aromatase inhibitor that blocks conversion of T to E2), BWSTT+TE, or BWSTT+TE+AN. Experiment 2b will be a dose- optimization study to identify the lowest E2 dose that enhances BWSTT-mediated neuromotor improvement. SCI animals will receive vehicle or BWSTT with low-, moderate-, or high-dose E2. Experiment 2c will be a comparative efficacy study in which SCI animals will receive vehicle, BWSTT or E2 (alone and in combination), or BWSTT+TE. We hypothesize that the functional and anatomical measures of neuromuscular plasticity will be most evident in SCI animals receiving BWSTT+TE, that E2 partially mediates neuromuscular plasticity resulting from BWSTT+TE, and that more robust neuromotor improvement will occur in response to BWSTT+TE versus BSWTT+E2. We anticipate that these experiments will provide the necessary data to establish preclinical proof-of-principle for our multimodal therapy. If successful, this will set the stage for translation of our preclinical findings to future clinical trials intended to hasten locomotor recovery and improve neuromuscular function in Veterans with SCI.